Abstract In mammalian cells the pH gradient between the organelles, cytoplasm and extracellular space is strictly regulated. Maintenance of pH homeostasis is crucial for the normal function of the cell and its organelles. In solid tumours, cells often suffer from hypoxia, deprivation of nutrients and acidic extracellular milieu as a result of inadequate vascularisation. Cancer cells are also known to suffer from other pH abnormalities. Defective acidification of intracellular organelles as well as a reversed pH gradient across the plasma membrane have been detected in numerous tumour tissues and cells. Aberrant secretion of lysosomal hydrolases, loss of cell polarity and increased expression of tumour-specific proteins are common phenotypic changes of cancer cells. In this study, secretion of cathepsin D, a lysosomal aspartic hydrolase, was shown to result from the acidification defect of cancer cells. In normal cells cathepsin D is sorted in the Golgi complex by mannose-6-phosphate receptors and transported via endosomes to lysosomes. In breast and colorectal cancer cells having abnormally neutral endosomes receptors were shown to accumulate in endosomes resulting in the aberrant secretion of newly synthesised cathepsin D from the cells. Carcinoembryonic antigen (CEA) is an oncofetal protein widely used as a tumour follow-up marker. It is normally expressed at low levels and is localised at the apical surface of epithelial cells via a glycosyl phosphatidyl inositol (GPI) anchor. In cancer cells the expression of CEA is increased and the protein is found over the entire surface of cells. In this study, the tumour microenvironmental factors, hypoxia and abnormal pH homeostasis, were shown to increase the expression of carcinoembryonic antigen in cancer cells. In addition, the absence of acidic organelles was shown to induce mistargeting of CEA to the basolateral membrane in polarised cells. The abnormally neutral Golgi was found to interfere with the complex formation of carcinoembryonic antigen, a phenomenon recently associated with the apical sorting of other GPI-anchored proteins. Altogether these results emphasise the role of tumour-related factors – altered pH homeostasis and hypoxia – in the phenotypic changes of cancer cells.

Cancer is still one of the most common causes of death world-wide and in parallel there is a need to update the repertoire of therapies that withstand resistance of recurrent cancers. Since the introduction of antibody therapies as anti-cancer pharmaceuticals, recognized as immunotherapy in health care, it has been an increasing field in cancer therapy, as a more targeted treatment compared to chemotherapy. Despite the great success, immunotherapy rely on parenteral administration, partly due to poor tissue penetration. If the treatment is administered intravenously, specialized personnel is required, in addition to that it can be inconvenient for the patient. Also, pharmaceuticals based on antibodies often require costly production steps which yields a high-priced treatment. To approach this problem, researchers have developed small affinity domains with the aim to increase tissue penetration while keeping a high specificity to its target. Albumin Binding Domain Derived Affinity Protein (ADAPT) is an example of a small affinity domain of only 7 kDa, which is based on albumin binding domain (ABD) from the streptococcal protein G. Recently, it was shown that the ADAPTs can be further engineered to bind albumin and another relevant target protein of interest simultaneously, which suggests a tolerable half-life in patient serum, alternative administration routes and lower production costs compared to antibody treatments. Furthermore, less side effects are expected due to higher specificity compared to chemotherapy. This work presents the characterization of novel ADAPT proteins that the target the cancer relatedproteins C-C motif ligand 7 (CCL7), vascular endothelial growth factor A (VEGF-A) and carcinoembryonic antigen related cell adhesion molecule 5 (CEACAM5). The new constructs were produced recombinantly in Escherichia coli (E. coli) and purified using affinity chromatography. Moreover, the results demonstrate bispecific binding with high affinity towards serum albumin and CCL7 and CEACAM5 respectively, while the ADAPT variants targeting VEGF-A remain to be further developed. Lastly, the importance of different amino acids for structural and binding properties of one CEACAM5 binder are stated. It reveals that the target binding relies on hydrophobic interactions which also can be connected to its poor structural attributes. Accordingly, this project adds new insights about the ADAPTs which can be useful in research towards future clinical applications aimed to improve cancer treatments.

Under senare år har målinriktad terapi varit ett växande område inom cancerterapi som en mer målinriktad behandling än kemoterapi. Dessa behandlingar baseras främst på antikroppsbaserade läkemedel som är ganska stora och komplexa, vilket ökar den totala kostnaden för behandlingen. Därför måste man hitta en alternativ metod för både upptäckt och behandling för att hjälpa patienterna. Små affinitetsdomäner har skapats med målet att förbättra vävnadspenetrationen och samtidigt upprätthålla en hög grad av målspecificitet, vilket leder till färre biverkningar. Ett av exemplen på detta är Albumin Binding Domain-Derived Affinity Protein (ADAPT). Det har baserats på en av de albuminbindande domänerna (ABD) i streptokockproteinet G, med en storlek på 6,5 kDa. Nyligen modifierades ADAPT ytterligare för att samtidigt binda albumin och ett annat relevant målprotein av intresse, vilket tyder på en längre halveringstid i patientserum och möjliggör utveckling av nyare och terapeutiska läkemedel. I detta projekt presenteras den fjärde generationen av ADAPT-biblioteket som utformats för att ha förbättrad stabilitet. Selektioner med fagdisplay utfördes mot tre målproteiner: carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5), en biomarkör för kolorektalcancer, epithelial cell adhesion molecule (EpCAM), en markör för flera gastrointestinala karcinom och trophoblast cell-surface antigen 2 (Trop2) som är överuttryckt i trippel-negativ bröstcancer. Resultaten visar bindning till CEACAM5, EpCAM och Trop2, vilket har visats med monoklonal fag-ELISA. Bindningsaffiniteten, sekundärstrukturen hos de utvalda bindarna och den bispecifika bindningen till serumalbumin återstår att utvärdera ytterligare. Projektet visar således att de ADAPTs som valts ut mot målen CEACAM5, EpCAM och Trop2 har en enorm potential för framtida kliniska tillämpningar som syftar till utveckling av diagnostik och terapi för dessa cancerbiomarkörer. / In recent years, targeted therapy has been a growing field of cancer therapy as a more targeted treatment than chemotherapy. These treatments are primarily based on antibody-based pharmaceuticals which are quite large and complex, increasing the overall cost of the treatment. Thus, an alternative method of both detection and treatment needs to be found to aid patients. Small affinity domains have been created with the goal of enhancing tissue penetration while maintaining a high level of target specificity, leading to fewer side effects. One of the examples for these is the Albumin Binding Domain-Derived Affinity Protein (ADAPT). It has been based on one of the albumin binding domains (ABD) of the streptococcal protein G, with a size of 6.5 kDa. Recently, the ADAPTs were further modified to simultaneously bind albumin and another pertinent target protein of interest, suggesting a longer half-life in patient serum, and enabling the development of newer therapeutics. This project presents the 4th generation of the ADAPT library designed to have improved stability. Phage display selections were performed against three target proteins: carcinoembryonic antigen- related cell adhesion molecule 5 (CEACAM5), a biomarker for colorectal cancer, epithelial cell adhesion molecule (EpCAM), a marker for several gastrointestinal carcinomas and trophoblast cell-surface antigen 2 (Trop2) which is overexpressed in triple-negative breast cancer. The results demonstrate binding towards CEACAM5, EpCAM and Trop2, which has been shown by monoclonal phage ELISA. The binding affinity, secondary structure of the selected binders and bispecific binding towards serum albumin remain to be further assessed. The project thus reveals that the ADAPTs selected against the targets CEACAM5, EpCAM and Trop2 present a massive potential for future clinical applications aimed towards development of diagnostics and therapeutics for these cancer biomarkers.